Role of Diversity and Recombination in the Emergence of Chilli Leaf Curl Virus.

Chilli leaf curl virus (ChiLCV), (Genus Begomovirus, family Geminiviridae) and associated satellites pose a serious threat to chilli production, worldwide. This study highlights the factors accountable for genetic diversity, recombination, and evolution of ChiLCV, and associated chilli leaf curl alphasatellite (ChiLCA) and chilli leaf curl betasatellite (ChiLCB). Phylogenetic analysis of complete genome (DNA-A) sequences of 132 ChiLCV isolates from five countries downloaded from NCBI database clustered into three major clades and showed high population diversity. The dN/dS ratio and Tajima D value of all viral DNA-A and associated betasatellite showed selective control on evolutionary relationships. Negative values of neutrality tests indicated purified selection and an excess of low-frequency polymorphism. Nucleotide diversity (π) for C4 and Rep genes was higher than other genes of ChiLCV with an average value of π = 18.37 × 10-2 and π = 17.52 × 10-2 respectively. A high number of mutations were detected in TrAP and Rep genes, while ChiLCB has a greater number of mutations than ChiLCA. In addition, significant recombination breakpoints were detected in all regions of ChiLCV genome, ChiLCB and, ChiLCA. Our findings indicate that ChiLCV has the potential for rapid evolution and adaptation to a range of geographic conditions and could be adopted to infect a wide range of crops, including diverse chilli cultivars.

Chilli leaf curl disease populations in India are highly recombinant, and rapidly segregated.

Capsicum annuum, a valuable spice and vegetable crop belonging to the Solanaceae family, is extensively grown across the Indian subcontinent. Chilli production is restricted by a begomoviral infection named as chilli leaf curl disease (ChiLCD) mainly in tropical and subtropical regions which leads to considerable economic losses, thus affecting chilli cultivation. Here, we studied the genetic diversity with structural evaluation of chilli leaf curl disease and satellite molecules infecting Chilli in India. We retrieved 121 reference sequences of ChiLCD including DNA-A, DNA-B, beta-satellite and alpha-satellites from GenBank reported from India. The population diversity and genetic variation were estimated through various parameters which decipher the four major groups of phylogenetic divergence for DNA-A and five groups of beta-satellite showing percentage similarity with isolates within and across India. Further, transitional and transversional bias for ORFs were observed highest in C4 and REn genes, respectively, and for DNA-A and DNA-B, these values were 1.07 and 1.22, respectively. The recombination breakpoints for DNA-A were estimated 49 majorly in V1, C1,C2 and C4 genome region and highest 22 breakpoints were determined for Rep (AC1) of ORFs, similarly 9 events for beta-satellite were found less around ßC1ORF. Moreover, the evolution and genetic variability were also contributed through parameters such as nucleotide substitution which were found within the range of RNA viruses for DNA-A, DNA-B, for all 6 ORFs (relaxed clock) and beta-satellite. Additionally, total numbers of mutations (η) for DNA-A, DNA-B, alpha-satellites and beta-satellites were 2505, 419, 807 and 1288 detected, respectively, while it was found 987 highest for Rep gene among all ORFs. Further, neutrality tests determine the dominant nature of population expansion and purifying selection for all the genes of begomovirus associated with ChiLCD and satellite molecules supporting conserved nature of gene. The combined Tajima's D and Fu and Li'S D* negative values in tests indicated that population are under purified selection and an excess of low-frequency polymorphism. Our analysis indicates the potential contribution of genetic mutations and recombination of ChiLCD which leads to rapid adaptation and evolution of begomovirus and its satellite molecules accelerating its host range and diversity within and across the Indian subcontinent. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03139-w.

Biology and Interaction of the Natural Occurrence of Distinct Monopartite Begomoviruses Associated With Satellites in Capsicum annum From India.

Chili (Capsicum annuum L.) is an important vegetable and spice crop of tropical and sub-tropical regions. Chili plants showing upward leaf curling, leaf crinkling, and leaf yellowing symptoms, collected from Sikar district of Rajasthan, India, were found to be associated with begomovirus and satellite molecules. The presence of virus was confirmed by PCR using virus-specific primer. The full-length genomic DNA-A of three begomovirus (MM-1, CS-1 and RV-1) and two satellites (MM-2 and MM-3) were cloned which was identified from single symptomatic chili plant. The genome organization of isolated three viruses is similar to those of other Old World monopartite begomoviruses. The comparison of the sequences and closest phylogenetic relationships for the begomoviruses, betasatellite and alphasatellite DNAs revealed that MM-1 was designated as DNA-A of Chili leaf curl virus (ChiLCV), CS-1 is considered to be a new distinct species of Tomato leaf curl Gujrat virus (ToLCGV) whereas RV-1 as a new strain of Cotton leaf curl Multan virus (CLCuMuV). The DNA-A component of ChiLCV showed 8.6%, ToLCGV of 16.6% and CLCuMuV of 7.7% average evolutionary divergence, concomitantly, the betasatellite and alphasatellite molecule had 9.9% and 5.9% overall sequence divergence, respectively. Interestingly, most of the begomoviruses were found to be intra-species recombinants. The dN/dS ratio and Tajima D value of all viral DNA-A component and their associated betasatellite showed their selective control on evolutionary relationships. The nucleotide substitution rates were determined for the DNA-A genomes of ChiLCV (7.22 × 10-4 substitutions site-1 year-1), CLCuMuV (1.49 × 10-4 substitutions site-1 year-1), ToLCGV (7.47 × 10-4 substitutions site-1 year-1), the genome of associated ChiLCB (4.20 × 10-4 substitutions site-1 year-1) and CLCuMuA (1.49 × 10-4 substitutions site-1 year-1). Agro-inoculation studies indicate that the presence of DNA betasatellite induce severe symptoms in N. benthamiana and chili, suggesting prerequisite association for typical disease development.

Identification, genetic diversity and recombination analysis of Watermelon Mosaic Virus isolates.

Watermelon mosaic virus (WMV) is an important virus causing adverse effects on cucurbits throughout the world. In this study, we recorded WMV infection in the watermelon (Citrullus lanatus)-growing area of Alwar and Sikar in districts of Rajasthan, India. The RT-PCR-based detection was performed to confirm the presence of WMV, by using potyvirus-degenerated coat protein primers. Further, the complete genome sequences of two WMV isolates were compared with previously reported genome sequences. The complete genome of each isolate was 10,030 nt long, excluding the poly-A tails. Phylogeny relationships of the WMV isolates in the present study revealed the presence of uneven evolutionary pressure among the different WMV viral genomic segments. The analysis revealed that all the WMV isolates were divided into three clusters and the Indian WMV isolates cluster together with the French isolate. Recombination analysis of WMV exhibited significant recombination hotspots in the P1, NIa-Pro and Nib-CP regions. Our finding highlights the importance of genetic variability and recombination analysis to provide a better understanding of WMV molecular diversity.

Identification of Chilli leaf curl virus and associated betasatellite infecting Osteospermum fruticosum in Rajasthan, India.

A monopartite begomovirus associated with betasatellite was identified from Osteospermum fruticosum (Cape Daisy) showing severe yellowing vein net symptoms in Rajasthan, India through molecular characterization. The DNA-A shared the highest nucleotide (96.61%) identity to Chilli leaf curl Ahmedabad virus (KM880103), while the betasatellite depicted the highest sequence similarity (99.28%) to Chilli leaf curl betasatellite (JF706231, 99.28%). Based on the sequence identity with other begomoviruses known to date, they were recognized as Chilli leaf curl virus (CDI, MH355641) and Chilli leaf curl betasatellite (CDB1, MH355642), respectively. Phylogenetic analysis showed that DNA-A (CD1) clustered with ChiLCV Goa (KP235539), whereas the betasatellite (CDB1) clustered with ChiLCB Jodhapur (JF70623). Recombination events were observed among the clades of ChiLCV, showing intragenic recombination in Rep (C1) and coat protein (V1/AV1) regions. To our knowledge, this is the first report of ChiLC begomovirus strain affecting O. fruticosum.